Thyroxine binding globulin (TBG) has been studied from several points of view. Physical properties of the molecule were investigated as affected by guanadinium chloride. The results indicated that the native molecule in the serum is in a metastable state since mild denaturation conditions lead to irreversible changes in molecular properties and loss of the binding site for thyroid hormone. Affinity labeling of TBG with bromacetyl thyroxine showed that the major amino acid interacting with this probe in the binding site was methionine. Minor amounts of lysine, tyrosine and histidine were also labeled. A theoretical schema for the calculation of the free thyroid hormones was developed utilizing known parameters of hormone binding in serum.